Growth differentiation factor-10

ABSTRACT

Growth differentiation factor-10 (GDF-10) polypeptide is disclosed as well as polynucleotides encoding GDF-10, vectors and host cells.

This application is a §371 of PCT/US94/11440 filed on Oct. 7, 1994,which is continuation-in-part of U.S. application Ser. No. 08/134,078filed on Oct. 8, 1993 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth factors and specifically to anew member of the transforming growth factor beta (TGF-β) superfamily,which is denoted, growth differentiation factor-10 (GDF-10).

2. Description of Related Art

The transforming growth factor β (TGF-β) superfamily encompasses a groupof structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325: 1-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs ((Weeks, et al.,Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPS,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198,1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

The proteins of the TGF-β family are initially synthesized as a largeprecursor protein which subsequently undergoes proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus. The C-terminal regions, or mat regions, of the proteins areall structurally related and the different family members can beclassified into distinct subgroups based on the extent of theirhomology. Although the homologies within particular subgroups range from70% to 90% amino acid sequence identity, the homologies betweensubgroups are significantly lower, generally ranging from only 20% to50%. In each case, the active species appears to be a disulfide-linkeddimer of C-terminal fragments. For most of the family members that havebeen studied, the homodimeric species has bee found to be biologicallyactive, but for other family members, like he inhibins (Ling, et al.,Nature, 321:779, 1986) and the TGF-βs (Cheifetz, et al., Cell, 48:409,1987), heterodimers have also been detected, and these appear to havedifferent biological properties than the respective homodimers.

Identification of new factors that are tissue-specific in theirexpression pattern will provide a greater understanding of that tissue'sdevelopment and function and allow development of effective diagnosticand therapeutic regimens.

SUMMARY OF THE INVENTION

The present invention provides a cell growth and differentiation factor,GDF-10, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving those involving uterine, nerve, bore, and adipose tissue.

Thus, in one embodiment, the invention provides a method for detecting acell proliferative disorder of uterine, nerve, or fat origin and whichis associated with GDF-10. In another embodiment, the invention providesa method for treating a cell proliferative disorder by suppressing orenhancing GDF-10 activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression of GDF-10 mRNA in adult tissues.

FIG. 2A and 2B show nucleotide and predicted amino acid sequence ofmurine GDF-10 (SEQ ID NO:4 and SEQ ID NO:5). Consensus N-glycosylationsignals are denoted by plain boxes.

FIG. 3A and 3B show the alignment of the C-terminal sequences of GDF-10(SEQ ID NO:5) with other members of the TGF-β superfamily (SEQ IDNO:7-24), respectively. The conserved cysteine residues are boxed.Dashes denote gap introduced in order to maximize alignment.

FIG. 4 shows amino acid homologies with different members of the TGF-βsuperfamily. Numbers represent percent amino acid identities betweeneach pair calculated from the first conserved cysteine to theC-terminus.

FIG. 5 shows an alignment of the C-terminal sequences of human (SEQ IDNO:26) (top lines) and murine (SEQ ID NO:25) (bottom lines) GDF-10.

FIG. 6 shows an autoradiogram of labeled secreted proteins synthesizedby 293 cells transfected with a pcDNAI vector into which the GDF-10 cDNAwas inserted in either the antisense (lanes 1 and 2) or sense (lanes 3and 4) orientation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a growth and differentiation factor,GDF-10 and a polynucleotide sequence encoding GDF-10. GDF-10 isexpressed at highest levels in uterus and fat and at lower levels inother tissues, such as brain. In one embodiment, the invention providesa method for detection of a cell proliferative disorder of uterine,nerve, or fat origin which is associated with GDF-10 expression. Inanother embodiment, the invention provides a method for treating a cellproliferative disorder by using an agent which suppresses or enhancesGDF-10 activity.

The TGF-β superfamily consists of multifunctional polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-10 protein of this invention and the members of the TGF-βfamily, indicates that GDF-10 is a new member of the family of growthand differentiation factors. Based on the known activities of many ofthe other members, it can be expected that GDF-10 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

The expression of GDF-10 in uterine and fat issue suggests a variety ofapplications using the polypeptide, polynucleotide, and antibodies ofthe invention, related to contraception, fertility, pregnancy, and cellproliferative diseases. Abnormally low levels of the factor my beindicative of impaired function in the uterus while abnormally highlevels may be indicative of hypertrophy, hyperplasia, or the presence ofectopic tissue. Hence, GDF-10 my be useful in detecting not only primaryand metastatic neoplasms of uterine origin but in detecting diseasessuch as endometriosis as well. In addition, GDF-10 may also be useful asan indicator of developmental anomalies in prenatal screeningprocedures.

Several members of the TGF-β superfamily possess activities suggestingpossible applications for the treatment of cell proliferative disorders,such as cancer. In particular, TGF-β has been shown to be potent growthinhibitor for a variety of cell types (Massague, Cell 49:437, 1987). MIShas been shown to in inhibit the growth of human endometrial carcinomatumors in nude amine (Donahoe, et al., Ann. Surg. 194:472, 1981), andinhibin α has been shown to suppress the development of tumors both inthe ovary an in the testis (Matzuk, et al., Nature, 360:313, 1992).GDF-10 may have similar activity and may therefore be useful as ananti-proliferative agent, such as for the treatment of endometrialcancer or endometriosis.

Many of the members of the TGF-β family are also important mediators oftissue repair. TGF-β has been shown to have marked effects on theformation of collagen and causes of striking angiogenic response in thenewborn mouse (Roberts, et al, Proc. Nat'l Acad. Sci., USA 83:4167,1986). The BMP's can induce new bone growth and are effective for thetreatment of fractures and other skeletal effects (Glowacki, et al.,Lancet, 1:959, 1981; Ferguson, et al., Clin. Orthoped. Relat Res.,227:265, 1988; Johnson, et al., Clin Orthoped Relat. Res., 230:257,1988). Based on the high degree of homology between GDF-10 and BMP-3,GDF-10 may have similar activities and may be useful in repair of tissueinjury caused by trauma or burns for example.

GDF-10 may play a role in regulation of the menstrual cycle orregulation of uterine function during pregnancy, and therefore, GDF-10,anti-GDF-10 antibodies, or antisense polynucleotides may be usefuleither in contraceptive regimens, in enhancing the success of in vitrofertilization procedures, or in preventing premature labor.

Certain members of this superfamily have expression patterns or possessactivities that relate to the function of the nervous system. Forexample, one family member, namely GDNF, has been shown to be a potentneurotrophic factor that can promote the survival of dopaminergicneurons (Lin, et al., Science, 260:1130). Another family member, namelydorsalin, is capable of promoting the differentiation of neural crestcells (Baster, et al., Cell, 73:687). The inhibins and activins havebeen shown to be expressed in the brain (Meunier, et al., Proc. Nat'lAcad. Sci., USA, 85:247, 1988; Sawchenko , et al., Nature, 334:615,1988), and activin has been shown to be capable of functioning as anerve cell survival molecule (Schubert, et al., Nature, 344:868, 1990).Another family member, namely GDF-1, is nervous system-specific in itsexpression pattern (Lee, Proc. Nat'l Acad. Sci., USA, 88:4250,1991), andcertain other family members, such as Vgr-1 (Lyons, et al., Proc. Nat'lAcad. Sci., USA, 86:4554, 1989; Jones et al., Development, 111:581,1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220, 1992), andBMP-4 (Jones, et al., Development 111:531, 1991), are also known to beexpressed in the nervous system. By analogy GDF-10 may have applicationsin the treatment of neurodegenerative diseases or in maintaining cellsor tissues in culture prior to transplantation.

The expression of GDF-10 in adipose tissue also raises the possibilityof applications for GDF-10 in the treatment of obesity or of disordersrelated to abnormal proliferation of adipocyte. In this regard, TGF-βhasbeen shown to be a potent inhibitor of adipocyte differentiation invitro (Ignotz and Massague, Proc. Natl. Acad. Sci., USA 82:8530, 1985).

The term “substantially pure” as used herein refers to GDF-10 which issubstantially free of other proteins, lipids carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify GDF-10 using standard techniques for protein purification.The substantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the GDF-10 polypeptidecan also be determined by amino-terminal amino acid sequence analysis.GDF-10 polypeptide includes functional fragments of the polypeptide, aslong as the activity of GDF-10 remains. Small peptides containing thebiological activity of GDF-10 are included in he invention.

The invention provides polynucleotides encoding the GDF-10 protein.These polynucleotides include DNA, cDNA and RNA sequences which encodeGDF-10. It is understood that all polynucleotides encoding all or aportion of GDF-10 are also included herein, as long as they encode apolypeptide with GDF-10 activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, GDF-10 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for GDF-10 also includesantisense sequences. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, a degenerate nucleotide sequences are included in theinvention as long as the amino acid sequence of GDF-10 polypeptideencoded by the nucleotide sequence is functionally unchanged.

Specifically disclosed herein is a cDNA sequence for GDF-10 which is2322 base pairs in length and contain an open reading frame beginningwith a methionine codon at nucleotide 126. The encoded polypeptide is476 amino acids in length with a molecular weight of about 52.5 kD, asdetermined by nucleotide sequence analysis. The GDF-10 sequence containsa core of hydrophobic amino acids near the N-terminus, suggestive of asignal sequence for secretion. GDF-10 contains four potentialN-glycosylation sites tasparagine residues 114, 152, 277, and 467.GDF-10 contains several potential proteolytic processing sites. Cleavagemost likely occurs following arginine 365, which would generate a maturefragment of DF-10 predicted to be 111 amino acids in length and have anunglycosylated molecular weight of about 12.6 kD, as determined bynucleotide sequence analysis. One skilled in the art can modify, orpartially or completely remove, the glycosyl groups from the GDF-10protein using standard techniques. Therefore the functional protein orfragments thereof of the invention includes glycosylated, partiallyglycosylated and unglycosylated species of GDF-10.

The C-terminal region of GDF-10 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β superfamily. The GDF-10 sequence contains most of the residuesthat are highly conserved in other family members. Among the knownfamily mammalian TGF-β family members, GDF-10 is most homologous toBMP-3 (83% sequence identity beginning with the first conserved cysteineresidue). GDF-10 also shows significant homology to BMP-3 (approximately30% sequence identity) in the pro-region of the molecule. Based on thesesequence comparisons, GDF-10 and BMP-3 appear to define a new subfamilywithin the larger superfamily.

Minor modifications of the recombinant GDF-10 primary amino acidsequence may result in proteins which have substantially equivalentactivity as compared to the GDF-10 polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the biological activity of GDF-10 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-10 bilogical activity.

The nucleotide sequence encoding the GDF-10 polypeptide of the inventionincludes the disclosed sequence and conservative variations thereof. Theterm “conservative variation” as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginin for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the GDF-10 polynucleotide of the invention is derived from amammalian organism, and most preferably from a mouse, rat, or human.Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogene us mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA ordenatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res. 9:879, 1981).

The development of specific DNA sequence, encoding GDF-10 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lamb a gt11, can be screenedindirectly for GDF-10 peptides having at least one epitope, usingantibodies specific for GDF-10. Such antibodies can be eitherpolyconally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-10 cDNA.

DNA sequences encoding GDF-10 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the GDF-10 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-10 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding GDF-10 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. Preferably, themature C-terminal region of DF-10 is expressed from a cDNA clonecontaining the entire coding sequence of GDF-10. Alternatively, theC-terminal portion of GDF-10 can be expressed as a fusion protein withthe pro- region of another member of the TGF-β family or co-expressedwith another pro- region (see for example, Hammonds, et al., Molec.Endocrin. 5:149, 1991; Gray, A., and Mason, A., Science, 247:1328,1990).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequent treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-10 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention includes antibodies immunoreactive with GDF-10 polypeptideor functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are mad from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). The term antibody as used in this inventionis meant to include intact molecules as well as fragments thereof, suchas Fab and F(ab′)₂ which are capable of binding an epitopic determinanton GDF-10.

The term “cell-proliferative disorder” denote malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically an genotypically. The term“cell-proliferative disorder” also includes situations in which anormally occurring process could be enhanced or suppressed for clinicalbenefit; an example of such a process would be fracture healing.Malignant cells (i.e. cancer) develop as a result of a multistepprocess. The GDF-10 polynucleotide that is an antisense molecule isuseful in treating malignancies of the various organ systems,particularly, for example, cells in uterine or adipose tissue.Essentially, any disorder which is etiologically linked to alteredexpression of GF-10 could be considered susceptible to treatment with aGDF-10 suppressing reagent. One such disorder is a malignant cellproliferative disorder, for example.

The invention provides a method for detecting a cell proliferativedisorder of uterine or adipose tissue which comprises contacting ananti-GDF-10 antibody with a cell suspected of having a GDF-10 associateddisorder and detecting binding to the antibody. The antibody reactivewith GDF-10 is labeled with a compound which allows detection of bindingto GDF-10. For purposes of the invention, an antibody specific forGDF-10 polypeptide may be used to detect the level of GDF-10 inbiological fluids and tissues. An specimen containing a detectableamount of antigen can be used. A preferred sample of this invention isuterine or fat tissue. The level of GDF-10 in the suspect cell can becompared with the level in a normal cell to determine whether thesubject has a GDF-10-associated cell proliferative disorder. Preferablythe subject is human.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising the polypeptideof the invention. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. These skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the antigen comprising a polypeptide of the invention for whichthe monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. Such dosages may vary, for example,depending on whether multiple injections are given, antigenic burden,and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylentriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹TI.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and 56Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of a GDF-10-associateddisease in a subject. Thus, for example, by measuring the increase ordecrease in the number of cells expressing antigen comprising apolypeptide of the invention or changes in the concentration of suchantigen present in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe GDF-10-associated disease is effective. The term “ameliorate”denotes a lessening of the detrimental effect of the GDF-10-associateddisease in the subject receiving therapy.

The present invention identifies a nucleotic be sequence that can beexpressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, where acell-proliferative disorder is associated with the expression of GDF-10,nucleic acid sequences that interfere with GDF-10 expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid and ribozymes to block translation of a specificGDF-10 mRNA, either by masking that mRNA with an antisense nucleic acidor by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1 990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target GDF-10-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes or inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative disorders which are mediated by GDF-10 protein. Suchtherapy would achieve its therapeutic effect by introduction of theGDF-10 antisense polynucleotide into cells having the proliferativedisorder. Delivery of antisense GDF-10 polynucleotide can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Especially preferred for therapeuticdelivery of antisense sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia or, preferably, an RNAvirus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a GDF-10 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, or example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the GDF-10 antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for GDF-10 antisense polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Due to the expression of GDF-10 primarily in uterine and adipose tissue,there are a variety of applications using the polypeptide,polynucleotide, and antibodies of the invention, related to these andother tissues. Such applications include treatment of cell proliferativedisorders involving these and other tissues, including bone. Inaddition, GDF-10 may be useful in various gene therapy procedures.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1

IDENTIFICATION AND ISOLATION OF A NOVEL TGF-β FAMILY MEMBER To identifynew members of the TGF-β superfamily, degenerate oligonucleotides weredesigned which corresponded to two conserved regions among the knownfamily members: one region downstream of the first conserved cysteineresidue and the other region spanning the invariant cysteine residuesnear the C-terminus. These primers were used for polymerase chainreactions on lung and brain cDNA followed by subcloning the PCR productsusing restriction sites placed at the 5′ ends of the primers, pickingindividual E. coli colonies carrying these subcloned inserts, and usinga combination of random sequencing and hybridization analysis toeliminate know members of the superfamily.

GDF-10 was identified from a mixture of PCR products obtained with theprimers:

NSC1: 5′- CCGGAATTCAA(G/A)GT(G/A/T/C)GA(T/C)TT(T/C)GC(G/A/T/C)GA       (T/C)AT(A/C/T)GG(G/A/T/C)TGG-3′ (SEQ ID NO:1) NSC2: 5′-CCGGMTTC(A/G)CA(G/A/T/C)GC(A/G)CA(G/A)CT(T/C)TC(G/A/T/C)       AC(G/A/T/C)GTCAT-3′ (SEQ ID NO:2) NSC3: 5′-CCGGAATTC(A/G)CA(G/A/T/C)GC(A/G)CA(G/A/T/C)GA(T/C)TC       (G/A/T/C)AC(G/A/T/C)GTCA-3′ (SEQ ID NO:3)

PCR using primers NSC1 with NSC2 or NSC1 with NSC3 was carried out withcDNA prepared from 0.25 μg of lung or brain mRNA for 35 cycles at 94° C.for 1 min, 50° C. for 2 min, and 72° C. for 2 min. PCR products ofapproximately 300 base pairs were digested with Eco RI, gel purified,and subcloned in the Bluescript vector (Stratagene, San Diego, Calif.).DNA was prepared from bacterial colonies carrying individual subclonesand sequenced. Of 11 clone that were sequenced, 9 corresponded to BMP-3,and two represented a novel sequence, which was designated GDF-10.

EXAMPLE 2 EXPRESSION PATTERN AND SEQUENCE OF GDF-10

To determine the expression pattern of GDF-10, RNA samples prepared froma variety of adult tissues were screened by Northern analysis. 2.5micrograms of twice polyA-selected RNA prepared from each tissue wereelectrophoresed on formaldehyde gels, blotted and probed with GDF-10. Asshown in FIG. 1, the GDF-10 probe detected an mRNA expressed at highestlevels in uterus, fat, and brain.

A murine uterus cDNA library consisting of 3×10⁶ recombinant phage wasconstructed in lambda ZAP II and screened with a probe derived from theGDF-10 PCR product. The entire nucleotide sequence of the longest of 7hybridizing clones is shown in FIG. 2. Consensus N-glycosylation signalsare denoted by plain boxes. Numbers indicate nucleotide positionrelative to the 5′ end. The 2322 bp sequence contains a long openreading frame beginning with a methionine codon at nucleotide 126 andpotentially encoding a protein 476 amino acids in length with amolecular weight of 52.5 kD. The predicted GDF-10 amino acid sequencecontains a hydrophobic N-terminal region, suggestive of a signalsequence for secretion, four potential N-linked glycosylation sites atasparagine residues 11 ,152, 277, and 467 and a putative proteolyticprocessing site at amino acid 365. Cleavage of the GDF-10 precursor atthis site would generate a mature GDF-10 protein 111 amino acids inlength with a predicted unglycosylated molecular weight of 12.6 kD.

The C-terminal region of GDF-10 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β superfamily (FIG. 3). FIG. 3 shows the alignment of the C-terminalsequences of GDF-10 with the corresponding regions of human GDF-1 (Lee,Proc. Natl. Acad. Sci. USA, 88:4250-4254, 1991), murine GDF-3 and GDF-9(McPherron and Lee, J. Biol. Chem. 268:3444, 1993), human BMP-2 and 4(Wozney, et al., Science, 242:1528-1534, 1988), human Vgr-1 (Celeste, etal., Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990), human OP-1(Ozkaynak, et al., EMBO J., 9:2085-2093,1990), human BMP-5 (Celeste, etal., Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990), human OP-2(Ozkaynak, et al., J. Biol. Chem., 267:25220-25227, 1992), human BMP-3(Wozney, et al., Science, 242:1528-1534, 1988), human MIS (Cate, et al.,Cell, 45:685-698, 1986), human inhibin alpha, βA, and, βB (Mason, etal., Biochem, Biophys. Res. Commun., 135:957-964, 1986), murine nodal(Zhou, et al., Nature, 361:543-547, 1993), human TGF-β1 (Derynck, etal., Nature, 316:701-705,1985), human TGF-β2 (deMartin, et al., EMBO J.,6:3673-3677, 1987), and human TGF-β3 (ten Dijke, et al., Proc. Natl.Acad. Sci. USA, 85:4715-4719,1988). The conserved cysteine residues areboxed. Dashes denote gaps introduced in order to maximize the alignment.

GDF-10 contains most of the residues that are highly conserved in otherfamily members, including the seven cysteine residues with theircharacteristic spacing.

FIG. 4 shows the amino acid homologies among the different members ofthe TGF-β superfamily. Numbers represent percent amino acid identitiescalculated from the first conserved cysteine to the C-terminus. In thisregion, GDF-10 is most homologous to BMP-3 (83% sequence identity).

EXAMPLE 3 ISOLATION OF HUMAN GDF-10

To isolate human GDF-10, a human uterus cDNA library consisting of16.2×10⁶ recombinant phage was constructed in lambda ZAP II and screenedwith a murine GDF-10 probe. From this library, 20 hybridizing cloneswere isolated. Partial nucleotide sequence analysis of the longest cloneshowed that human and murine GDF-10 are highly homologous; the predictedamino acid sequences are 97% identical beginning with the firstconserved cysteinie residue following the predicted cleavage site (FIG.5).

EXAMPLE 4 SECRETION OF GDF-10 BY MAMMALIAN CELLS

To determine whether GDF-10 is secreted by mammalian cells, the GDF-10cDNA was cloned into the pcDNAI expression vector and transfected into293 cells. Following DNA transfection, the cells were metabolicallylabeled with a mixture of [³⁵S]-cysteine and [³⁵S]-methionine, andlabeled secreted proteins were analyzed by SDS-polyacrylamide gelelectrophoresis. As show in FIG. 6, additional bands were detected incells transfected with a sense GDF-10 construct compared to an antisensecontrol construct. The presence of multiple protein species most likelyindicates that 293 cells are capable of proteolytically processingGDF-10. Hence, these data suggest that GDF-10 is secreted by these cellsand that GDF-10 is cleaved, as predicted from the cDNA sequence.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

26 36 base pairs nucleic acid single linear DNA (genomic) unknown NSC1CDS 1..36 1 CCGGAATTCA ARGTNGAYTT YGCNGAYATH GGNTGG 36 33 base pairsnucleic acid single linear DNA (genomic) unknown NSC2 CDS 1..33 2CCGGAATTCR CANGCRCARC TYTCNACNGT CAT 33 33 base pairs nucleic acidsingle linear DNA (genomic) unknown NSC3 CDS 1..33 3 CCGGAATTCRCANGCRCANG AYTCNACNGT CAT 33 2322 base pairs nucleic acid single linearDNA (genomic) unknown Murine GDF-10 CDS 126..1553 4 TGGGGTCATCCGGGCTGTCC GAGTCCCACA GGGACAACTC CAGCCGCGGA CGAGGTGCAC 60 AGCCAACACTGAGCCCTCCT TGTCTGTTCT CCTGGGCTCA GACCCTTCAC CACCGTTACT 120 CAGCC ATG GCTCCA GGT CCT GCT CGG ATC AGC TTG GGG TCC CAG CTG 167 Met Ala Pro Gly ProAla Arg Ile Ser Leu Gly Ser Gln Leu 1 5 10 CTG CCC ATG GTG CCG CTG CTCCTG CTG CTG CGG GGC GCA GGC TGC GGC 215 Leu Pro Met Val Pro Leu Leu LeuLeu Leu Arg Gly Ala Gly Cys Gly 15 20 25 30 CAC AGG GGC CCC TCA TGG TCCTCA TTG CCC TCG GCA GCT GCC GGT CTG 263 His Arg Gly Pro Ser Trp Ser SerLeu Pro Ser Ala Ala Ala Gly Leu 35 40 45 CAG GGG GAC AGG GAC TCC CAG CAGTCA CCC GGG GAC GCA GCA GCC GCT 311 Gln Gly Asp Arg Asp Ser Gln Gln SerPro Gly Asp Ala Ala Ala Ala 50 55 60 CTG GGC CCA GGC GCC CAG GAC ATG GTCGCT ATC CAC ATG CTC AGG CTC 359 Leu Gly Pro Gly Ala Gln Asp Met Val AlaIle His Met Leu Arg Leu 65 70 75 TAT GAG AAG TAC AAC CGA AGA GGT GCT CCACCG GGA GGA GGC AAC ACC 407 Tyr Glu Lys Tyr Asn Arg Arg Gly Ala Pro ProGly Gly Gly Asn Thr 80 85 90 GTC CGA AGC TTC CGT GCC CGG CTG GAA ATG ATCGAC CAA AAG CCT GTG 455 Val Arg Ser Phe Arg Ala Arg Leu Glu Met Ile AspGln Lys Pro Val 95 100 105 110 TAT TTC TTC AAC TTG ACT TCC ATG CAA GACTCA GAA ATG ATC CTC ACA 503 Tyr Phe Phe Asn Leu Thr Ser Met Gln Asp SerGlu Met Ile Leu Thr 115 120 125 GCC GCC TTC CAC TTC TAC TCA GAA CCT CCACGG TGG CCC CGG GCT GGT 551 Ala Ala Phe His Phe Tyr Ser Glu Pro Pro ArgTrp Pro Arg Ala Gly 130 135 140 GAG GTA TTC TGC AAG CCC CGA GCT AAG AACGCA TCC TGC CGC CTC CTG 599 Glu Val Phe Cys Lys Pro Arg Ala Lys Asn AlaSer Cys Arg Leu Leu 145 150 155 ACC CCA GGG CTG CCT GCA CGC TTG CAC CTAATC TTC CGC AGT CTT TCC 647 Thr Pro Gly Leu Pro Ala Arg Leu His Leu IlePhe Arg Ser Leu Ser 160 165 170 CAG AAC ACC GCC ACT CAG GGG CTG CTC CGCGGG GCC ATG GCC CTG ACG 695 Gln Asn Thr Ala Thr Gln Gly Leu Leu Arg GlyAla Met Ala Leu Thr 175 180 185 190 CCT CCA CCA CGT GGC CTG TGG CAG GCCAAG GAC ATC TCC TCA ATC ATC 743 Pro Pro Pro Arg Gly Leu Trp Gln Ala LysAsp Ile Ser Ser Ile Ile 195 200 205 AAG GCT GCC CGA AGG GAT GGA GAG CTGCTT CTC TCT GCT CAG CTG GAT 791 Lys Ala Ala Arg Arg Asp Gly Glu Leu LeuLeu Ser Ala Gln Leu Asp 210 215 220 ACT GGG GAG AAG GAC CCC GGA GTG CCACGG CCC AGT TCC CAC ATG CCC 839 Thr Gly Glu Lys Asp Pro Gly Val Pro ArgPro Ser Ser His Met Pro 225 230 235 TAT ATC CTT GTC TAC GCC AAT GAC CTGGCC ATC TCC GAA CCC AAC AGT 887 Tyr Ile Leu Val Tyr Ala Asn Asp Leu AlaIle Ser Glu Pro Asn Ser 240 245 250 GTA GCA GTG TCG CTA CAG AGA TAC GACCCA TTT CCA GCT GGA GAC TTT 935 Val Ala Val Ser Leu Gln Arg Tyr Asp ProPhe Pro Ala Gly Asp Phe 255 260 265 270 GAG CCT GGA GCA GCC CCC AAC AGCTCA GCT GAT CCC CGC GTG CGC AGG 983 Glu Pro Gly Ala Ala Pro Asn Ser SerAla Asp Pro Arg Val Arg Arg 275 280 285 GCG GCT CAG GTG TCA AAA CCC CTGCAA GAC AAT GAA CTG CCG GGG CTG 1031 Ala Ala Gln Val Ser Lys Pro Leu GlnAsp Asn Glu Leu Pro Gly Leu 290 295 300 GAT GAA AGA CCA GCG CCT GCC CTGCAT GCC CAG AAT TTC CAC AAG CAC 1079 Asp Glu Arg Pro Ala Pro Ala Leu HisAla Gln Asn Phe His Lys His 305 310 315 GAG TTC TGG TCC AGT CCT TTC CGGGCA CTG AAA CCC CGC ACG GCG CGC 1127 Glu Phe Trp Ser Ser Pro Phe Arg AlaLeu Lys Pro Arg Thr Ala Arg 320 325 330 AAA GAC CGC AAG AAG AAG GAC CAGGAC ACA TTC ACC GCC GCC TCC TCT 1175 Lys Asp Arg Lys Lys Lys Asp Gln AspThr Phe Thr Ala Ala Ser Ser 335 340 345 350 CAG GTG CTG GAC TTT GAC GAGAAG ACG ATG CAG AAA GCC AGG AGG CGG 1223 Gln Val Leu Asp Phe Asp Glu LysThr Met Gln Lys Ala Arg Arg Arg 355 360 365 CAG TGG GAT GAG CCC CGG GTCTGC TCC AGG AGG TAC CTG AAG GTG GAT 1271 Gln Trp Asp Glu Pro Arg Val CysSer Arg Arg Tyr Leu Lys Val Asp 370 375 380 TTT GCA GAC ATC GGG TGG AATGAA TGG ATC ATC TCT CCC AAA TCC TTT 1319 Phe Ala Asp Ile Gly Trp Asn GluTrp Ile Ile Ser Pro Lys Ser Phe 385 390 395 GAC GCC TAC TAC TGT GCT GGGGCC TGC GAG TTC CCC ATG CCC AAG ATT 1367 Asp Ala Tyr Tyr Cys Ala Gly AlaCys Glu Phe Pro Met Pro Lys Ile 400 405 410 GTC CGC CCA TCC AAC CAT GCCACC ATC CAG AGC ATC GTC AGA GCT GTG 1415 Val Arg Pro Ser Asn His Ala ThrIle Gln Ser Ile Val Arg Ala Val 415 420 425 430 GGC ATT GTC CCT GGC ATCCCA GAG CCA TGC TGT GTT CCA GAC AAG ATG 1463 Gly Ile Val Pro Gly Ile ProGlu Pro Cys Cys Val Pro Asp Lys Met 435 440 445 AAC TCC CTT GGA GTC CTTTTC CTG GAT GAA AAT CGG AAT GCG GTT CTG 1511 Asn Ser Leu Gly Val Leu PheLeu Asp Glu Asn Arg Asn Ala Val Leu 450 455 460 AAG GTG TAC CCC AAT ATGTCC GTA GAG ACC TGT GCC TGT CGG 1553 Lys Val Tyr Pro Asn Met Ser Val GluThr Cys Ala Cys Arg 465 470 475 TAAGATGGCT TCAAGATAGA AGACAGACCTGCTTCATCCC TGCCCTGCAG AGTGGCAATC 1613 TTGGAGCCAG GGACTTGACT CGGGGAGGTTCCAGGTGCTA GACAGAGCTT ACAGGCAGCC 1673 CTGCTGGGAC CAAGAAAGAT CTGCCCACCACATCGCAATT CTTCAGTTCT TCCGTGCTGG 1733 TGGTAGCTCT GTAAAGACGT GTTGAGTTCCTGGAAGAAAT CTGGAATTAA CTGTGGTCTG 1793 CAATTTGCCC ATCATCCCTG CCCACACTTTTCAAGGCCTA GAAATAACGT GTGTCCTCAA 1853 ATGTCAACTC CAGGCATTTG TCCTCTCAAAACCTAGAAAG ACTATGCAAA TCTTGGGGTA 1913 CTCCCCCCCC CCATGGCAGT TTAAATGCTGTTTTAAAACC CTCAGGCTGC ATTCTAGAAA 1973 CAGGGCCTAA CCCATGGCAC GAGTGAGTATTTTCTCTTAC GTTTCACTAC ACGTGCTTTT 2033 ATACATGCAG TATGCACATG TAATCACGGTTGATTTCTTC TTTTAATATA TGTATTTCTA 2093 TTTCAAAGCA AAACGGAGAG AGTCGATCCCATCCCCTGCA GAGGTAATAA TGCAAGTTAG 2153 GTGTGGGTTG TCTAAGCATG TGTATGGAAATAATACATAC AGTAATATGC TGGAATACTA 2213 AAAAAGTAAC CAAGATTTTA TATTTTTGTAAATTATACTT TGTATACTGT AGATTGTGAG 2273 TGTTCTGTGT TTTTATGGAA AGCTAATAAATTAAAGGTGC GGAGGTATC 2322 476 amino acids amino acid linear proteinunknown 5 Met Ala Pro Gly Pro Ala Arg Ile Ser Leu Gly Ser Gln Leu LeuPro 1 5 10 15 Met Val Pro Leu Leu Leu Leu Leu Arg Gly Ala Gly Cys GlyHis Arg 20 25 30 Gly Pro Ser Trp Ser Ser Leu Pro Ser Ala Ala Ala Gly LeuGln Gly 35 40 45 Asp Arg Asp Ser Gln Gln Ser Pro Gly Asp Ala Ala Ala AlaLeu Gly 50 55 60 Pro Gly Ala Gln Asp Met Val Ala Ile His Met Leu Arg LeuTyr Glu 65 70 75 80 Lys Tyr Asn Arg Arg Gly Ala Pro Pro Gly Gly Gly AsnThr Val Arg 85 90 95 Ser Phe Arg Ala Arg Leu Glu Met Ile Asp Gln Lys ProVal Tyr Phe 100 105 110 Phe Asn Leu Thr Ser Met Gln Asp Ser Glu Met IleLeu Thr Ala Ala 115 120 125 Phe His Phe Tyr Ser Glu Pro Pro Arg Trp ProArg Ala Gly Glu Val 130 135 140 Phe Cys Lys Pro Arg Ala Lys Asn Ala SerCys Arg Leu Leu Thr Pro 145 150 155 160 Gly Leu Pro Ala Arg Leu His LeuIle Phe Arg Ser Leu Ser Gln Asn 165 170 175 Thr Ala Thr Gln Gly Leu LeuArg Gly Ala Met Ala Leu Thr Pro Pro 180 185 190 Pro Arg Gly Leu Trp GlnAla Lys Asp Ile Ser Ser Ile Ile Lys Ala 195 200 205 Ala Arg Arg Asp GlyGlu Leu Leu Leu Ser Ala Gln Leu Asp Thr Gly 210 215 220 Glu Lys Asp ProGly Val Pro Arg Pro Ser Ser His Met Pro Tyr Ile 225 230 235 240 Leu ValTyr Ala Asn Asp Leu Ala Ile Ser Glu Pro Asn Ser Val Ala 245 250 255 ValSer Leu Gln Arg Tyr Asp Pro Phe Pro Ala Gly Asp Phe Glu Pro 260 265 270Gly Ala Ala Pro Asn Ser Ser Ala Asp Pro Arg Val Arg Arg Ala Ala 275 280285 Gln Val Ser Lys Pro Leu Gln Asp Asn Glu Leu Pro Gly Leu Asp Glu 290295 300 Arg Pro Ala Pro Ala Leu His Ala Gln Asn Phe His Lys His Glu Phe305 310 315 320 Trp Ser Ser Pro Phe Arg Ala Leu Lys Pro Arg Thr Ala ArgLys Asp 325 330 335 Arg Lys Lys Lys Asp Gln Asp Thr Phe Thr Ala Ala SerSer Gln Val 340 345 350 Leu Asp Phe Asp Glu Lys Thr Met Gln Lys Ala ArgArg Arg Gln Trp 355 360 365 Asp Glu Pro Arg Val Cys Ser Arg Arg Tyr LeuLys Val Asp Phe Ala 370 375 380 Asp Ile Gly Trp Asn Glu Trp Ile Ile SerPro Lys Ser Phe Asp Ala 385 390 395 400 Tyr Tyr Cys Ala Gly Ala Cys GluPhe Pro Met Pro Lys Ile Val Arg 405 410 415 Pro Ser Asn His Ala Thr IleGln Ser Ile Val Arg Ala Val Gly Ile 420 425 430 Val Pro Gly Ile Pro GluPro Cys Cys Val Pro Asp Lys Met Asn Ser 435 440 445 Leu Gly Val Leu PheLeu Asp Glu Asn Arg Asn Ala Val Leu Lys Val 450 455 460 Tyr Pro Asn MetSer Val Glu Thr Cys Ala Cys Arg 465 470 475 120 amino acids amino acidsingle linear protein unknown GDF-10 Protein 1..120 6 Glu Lys Ser MetGln Lys Ala Arg Arg Arg Gln Trp Asp Glu Pro Arg 1 5 10 15 Val Cys SerArg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp 20 25 30 Asn Glu TrpIle Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala 35 40 45 Gly Ala CysGlu Phe Pro Met Pro Lys Ile Val Arg Pro Ser Asn His 50 55 60 Ala Thr IleGln Ser Ile Val Arg Ala Val Gly Ile Val Pro Gly Ile 65 70 75 80 Pro GluPro Cys Cys Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu 85 90 95 Phe LeuAsp Glu Asn Arg Asn Ala Val Leu Lys Val Tyr Pro Asn Met 100 105 110 SerVal Glu Thr Cys Ala Cys Arg 115 120 123 amino acids amino acid singlelinear protein unknown GDF-1 Protein 1..123 7 Arg Pro Arg Arg Asp AlaGlu Pro Val Leu Gly Gly Gly Pro Gly Gly 1 5 10 15 Ala Cys Arg Ala ArgArg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp 20 25 30 His Arg Trp Val IleAla Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gln 35 40 45 Gly Gln Cys Ala LeuPro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro 50 55 60 Ala Leu Asn His AlaVal Leu Arg Ala Leu Met His Ala Ala Ala Pro 65 70 75 80 Gly Ala Ala AspLeu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile 85 90 95 Ser Val Leu PhePhe Asp Asn Ser Asp Asn Val Val Leu Arg Gln Tyr 100 105 110 Glu Asp MetVal Val Asp Glu Cys Gly Cys Arg 115 120 118 amino acids amino acidsingle linear protein unknown GDF-3 Protein 1..118 8 Arg Lys Arg Arg AlaAla Ile Ser Val Pro Lys Gly Phe Cys Arg Asn 1 5 10 15 Phe Cys His ArgHis Gln Leu Phe Ile Asn Phe Gln Asp Leu Gly Trp 20 25 30 His Lys Trp ValIle Ala Pro Lys Gly Phe Met Ala Asn Tyr Cys His 35 40 45 Gly Glu Cys ProPhe Ser Met Thr Thr Tyr Leu Asn Ser Ser Asn Tyr 50 55 60 Ala Phe Met GlnAla Leu Met His Met Ala Asp Pro Lys Val Pro Lys 65 70 75 80 Ala Val CysVal Pro Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Gln 85 90 95 Asp Ser AspLys Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val 100 105 110 Asp GluCys Gly Cys Gly 115 119 amino acids amino acid single linear proteinunknown GDF-9 Protein 1..119 9 Ser Phe Asn Leu Ser Glu Tyr Phe Lys GlnPhe Leu Phe Pro Gln Asn 1 5 10 15 Glu Cys Glu Leu His Asp Phe Arg LeuSer Phe Ser Gln Leu Lys Trp 20 25 30 Asp Asn Trp Ile Val Ala Pro His ArgTyr Asn Pro Arg Tyr Cys Lys 35 40 45 Gly Asp Cys Pro Arg Ala Val Arg HisArg Tyr Gly Ser Pro Val His 50 55 60 Thr Met Val Gln Asn Ile Ile Tyr GluLys Leu Asp Pro Ser Val Pro 65 70 75 80 Arg Pro Ser Cys Val Pro Gly LysTyr Ser Pro Leu Ser Val Leu Thr 85 90 95 Ile Glu Pro Asp Gly Ser Ile AlaTyr Lys Glu Tyr Glu Asp Met Ile 100 105 110 Ala Thr Arg Cys Thr Cys Arg115 118 amino acids amino acid single linear protein unknown BMP-2Protein 1..118 10 Arg Glu Lys Arg Gln Ala Lys His Lys Gln Arg Lys ArgLeu Lys Ser 1 5 10 15 Ser Cys Lys Arg His Pro Leu Tyr Val Asp Phe SerAsp Val Gly Trp 20 25 30 Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His AlaPhe Tyr Cys His 35 40 45 Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu AsnSer Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn SerLys Ile Pro Lys 65 70 75 80 Ala Cys Cys Val Pro Thr Glu Leu Ser Ala IleSer Met Leu Tyr Leu 85 90 95 Asp Glu Asn Glu Lys Val Val Leu Lys Asn TyrGln Asp Met Val Val 100 105 110 Glu Gly Cys Gly Cys Arg 115 118 aminoacids amino acid single linear protein unknown BMP-4 Protein 1..118 11Lys Arg Ser Pro Lys His His Ser Gln Arg Ala Arg Lys Lys Asn Lys 1 5 1015 Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp 20 2530 Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His 35 4045 Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His 50 5560 Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys 65 7075 80 Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu 8590 95 Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val100 105 110 Glu Gly Cys Gly Cys Arg 115 119 amino acids amino acidsingle linear protein unknown Vgr-1 Protein 1..119 12 Ser Arg Gly SerGly Ser Ser Asp Tyr Asn Gly Ser Glu Leu Lys Thr 1 5 10 15 Ala Cys LysLys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp 20 25 30 Gln Asp TrpIle Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp 35 40 45 Gly Glu CysSer Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 50 55 60 Ala Ile ValGln Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro 65 70 75 80 Lys ProCys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr 85 90 95 Phe AspAsp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val 100 105 110 ValArg Ala Cys Gly Cys His 115 119 amino acids amino acid single linearprotein unknown OP-1 Protein 1..119 13 Leu Arg Met Ala Asn Val Ala GluAsn Ser Ser Ser Asp Gln Arg Gln 1 5 10 15 Ala Cys Lys Lys His Glu LeuTyr Val Ser Phe Arg Asp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala ProGlu Gly Tyr Ala Ala Tyr Tyr Cys Glu 35 40 45 Gly Glu Cys Ala Phe Pro LeuAsn Ser Tyr Met Asn Ala Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu ValHis Phe Ile Asn Pro Glu Thr Val Pro 65 70 75 80 Lys Pro Cys Cys Ala ProThr Gln Leu Asn Ala Ile Ser Val Leu Tyr 85 90 95 Phe Asp Asp Ser Ser AsnVal Ile Leu Lys Lys Tyr Arg Asn Met Val 100 105 110 Val Arg Ala Cys GlyCys His 115 119 amino acids amino acid single linear protein unknownBMP-5 Protein 1..119 14 Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr SerGlu Gln Lys Gln 1 5 10 15 Ala Cys Lys Lys His Glu Leu Tyr Val Ser PheArg Asp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr AlaAla Phe Tyr Cys Asp 35 40 45 Gly Glu Cys Ser Phe Pro Leu Asn Ala His MetAsn Ala Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val His Leu Met PhePro Asp His Val Pro 65 70 75 80 Lys Pro Cys Cys Ala Pro Thr Lys Leu AsnAla Ile Ser Val Leu Tyr 85 90 95 Phe Asp Asp Ser Ser Asn Val Ile Leu LysLys Tyr Arg Asn Met Val 100 105 110 Val Arg Ser Cys Gly Cys His 115 119amino acids amino acid single linear protein unknown OP-2 Protein 1..11915 Arg Leu Pro Gly Ile Phe Asp Asp Val His Gly Ser His Gly Arg Gln 1 510 15 Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp 2025 30 Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Glu 3540 45 Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His 5055 60 Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro 6570 75 80 Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr85 90 95 Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys Ala Arg Asn Met Val100 105 110 Val Lys Ala Cys Gly Cys His 115 120 amino acids amino acidsingle linear protein unknown BMP-3 Protein 1..120 16 Glu Gln Thr LeuLys Lys Ala Arg Arg Lys Gln Trp Ile Glu Pro Arg 1 5 10 15 Asn Cys AlaArg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp 20 25 30 Ser Glu TrpIle Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser 35 40 45 Gly Ala CysGln Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His 50 55 60 Ala Thr IleGln Ser Ile Val Arg Ala Val Gly Val Val Pro Gly Ile 65 70 75 80 Pro GluPro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser Ile Leu 85 90 95 Phe PheAsp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 100 105 110 ThrVal Glu Ser Cys Ala Cys Arg 115 120 116 amino acids amino acid singlelinear protein unknown MIS Protein 1..116 17 Gly Pro Gly Arg Ala Gln ArgSer Ala Gly Ala Thr Ala Ala Asp Gly 1 5 10 15 Pro Cys Ala Leu Arg GluLeu Ser Val Asp Leu Arg Ala Glu Arg Ser 20 25 30 Val Leu Ile Pro Glu ThrTyr Gln Ala Asn Asn Cys Gln Gly Val Cys 35 40 45 Gly Trp Pro Gln Ser AspArg Asn Pro Arg Tyr Gly Asn His Val Val 50 55 60 Leu Leu Leu Lys Met GlnAla Arg Gly Ala Ala Leu Ala Arg Pro Pro 65 70 75 80 Cys Cys Val Pro ThrAla Tyr Ala Gly Lys Leu Leu Ile Ser Leu Ser 85 90 95 Glu Glu Arg Ile SerAla His His Val Pro Asn Met Val Ala Thr Glu 100 105 110 Cys Gly Cys Arg115 122 amino acids amino acid single linear protein unknownInhibin-alpha Protein 1..122 18 Ala Leu Arg Leu Leu Gln Arg Pro Pro GluGlu Pro Ala Ala His Ala 1 5 10 15 Asn Cys His Arg Val Ala Leu Asn IleSer Phe Gln Glu Leu Gly Trp 20 25 30 Glu Arg Trp Ile Val Tyr Pro Pro SerPhe Ile Phe His Tyr Cys His 35 40 45 Gly Gly Cys Gly Leu His Ile Pro ProAsn Leu Ser Leu Pro Val Pro 50 55 60 Gly Ala Pro Pro Thr Pro Ala Gln ProTyr Ser Leu Leu Pro Gly Ala 65 70 75 80 Gln Pro Cys Cys Ala Ala Leu ProGly Thr Met Arg Pro Leu His Val 85 90 95 Arg Thr Thr Ser Asp Gly Gly TyrSer Phe Lys Tyr Glu Thr Val Pro 100 105 110 Asn Leu Leu Thr Gln His CysAla Cys Ile 115 120 121 amino acids amino acid single linear proteinunknown Inhibin-beta-A Protein 1..121 19 Arg Arg Arg Arg Arg Gly Leu GluCys Asp Gly Lys Val Asn Ile Cys 1 5 10 15 Cys Lys Lys Gln Phe Phe ValSer Phe Lys Asp Ile Gly Trp Asn Asp 20 25 30 Trp Ile Ile Ala Pro Ser GlyTyr His Ala Asn Tyr Cys Glu Gly Glu 35 40 45 Cys Pro Ser His Ile Ala GlyThr Ser Gly Ser Ser Leu Ser Phe His 50 55 60 Ser Thr Val Ile Asn His TyrArg Met Arg Gly His Ser Pro Phe Ala 65 70 75 80 Asn Leu Lys Ser Cys CysVal Pro Thr Lys Leu Arg Pro Met Ser Met 85 90 95 Leu Tyr Tyr Asp Asp GlyGln Asn Ile Ile Lys Lys Asp Ile Gln Asn 100 105 110 Met Ile Val Glu GluCys Gly Cys Ser 115 120 120 amino acids amino acid single linear proteinunknown Inhibin-beta-B Protein 1..120 20 Arg Ile Arg Lys Arg Gly Leu GluCys Asp Gly Arg Thr Asn Leu Cys 1 5 10 15 Cys Arg Gln Gln Phe Phe IleAsp Phe Arg Leu Ile Gly Trp Asn Asp 20 25 30 Trp Ile Ile Ala Pro Thr GlyTyr Tyr Gly Asn Tyr Cys Glu Gly Ser 35 40 45 Cys Pro Ala Tyr Leu Ala GlyVal Pro Gly Ser Ala Ser Ser Phe His 50 55 60 Thr Ala Val Val Asn Gln TyrArg Met Arg Gly Leu Asn Pro Gly Thr 65 70 75 80 Val Asn Ser Cys Cys IlePro Thr Lys Leu Ser Thr Met Ser Met Leu 85 90 95 Tyr Phe Asp Asp Glu TyrAsn Ile Val Lys Arg Asp Val Pro Asn Met 100 105 110 Ile Val Glu Glu CysGly Cys Ala 115 120 118 amino acids amino acid single linear proteinunknown Nodal Protein 1..118 21 Gly Trp Gly Arg Arg Gln Arg Arg His HisLeu Pro Asp Arg Ser Gln 1 5 10 15 Leu Cys Arg Arg Val Lys Phe Gln ValAsp Phe Asn Leu Ile Gly Trp 20 25 30 Gly Ser Trp Ile Ile Tyr Pro Lys GlnTyr Asn Ala Tyr Arg Cys Glu 35 40 45 Gly Glu Cys Pro Asn Pro Val Gly GluGlu Phe His Pro Thr Asn His 50 55 60 Ala Tyr Ile Gln Ser Leu Leu Lys ArgTyr Gln Pro His Arg Val Pro 65 70 75 80 Ser Thr Cys Cys Ala Pro Val LysThr Lys Pro Leu Ser Met Leu Tyr 85 90 95 Val Asp Asn Gly Arg Val Leu LeuGlu His His Lys Asp Met Ile Val 100 105 110 Glu Glu Cys Gly Cys Leu 115114 amino acids amino acid single linear protein unknown TGF-beta-1Protein 1..114 22 Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser ThrGlu Lys Asn 1 5 10 15 Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg LysAsp Leu Gly Trp 20 25 30 Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala AsnPhe Cys Leu Gly 35 40 45 Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln TyrSer Lys Val Leu 50 55 60 Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser AlaAla Pro Cys Cys 65 70 75 80 Val Pro Gln Ala Leu Glu Pro Leu Pro Ile ValTyr Tyr Val Gly Arg 85 90 95 Lys Pro Lys Val Glu Gln Leu Ser Asn Met IleVal Arg Ser Cys Lys 100 105 110 Cys Ser 114 amino acids amino acidsingle linear protein unknown TGF-beta-2 Protein 1..114 23 Lys Arg AlaLeu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn 1 5 10 15 Cys CysLeu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp 20 25 30 Lys TrpIle His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly 35 40 45 Ala CysPro Tyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg Val Leu 50 55 60 Ser LeuTyr Asn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 65 70 75 80 ValSer Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys 85 90 95 ThrPro Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys 100 105 110Cys Ser 114 amino acids amino acid single linear protein unknownTGF-beta-3 Protein 1..114 24 Lys Arg Ala Leu Asp Thr Asn Tyr Cys Phe ArgAsn Leu Glu Glu Asn 1 5 10 15 Cys Cys Val Arg Pro Leu Tyr Ile Asp PheArg Gln Asp Leu Gly Trp 20 25 30 Lys Trp Val His Glu Pro Lys Gly Tyr TyrAla Asn Phe Cys Ser Gly 35 40 45 Pro Cys Pro Tyr Leu Arg Ser Ala Asp ThrThr His Ser Thr Val Leu 50 55 60 Gly Leu Tyr Asn Thr Leu Asn Pro Glu AlaSer Ala Ser Pro Cys Cys 65 70 75 80 Val Pro Gln Asp Leu Glu Pro Leu ThrIle Leu Tyr Tyr Val Gly Arg 85 90 95 Thr Pro Lys Val Glu Gln Leu Ser AsnMet Val Val Lys Ser Cys Lys 100 105 110 Cys Ser 115 amino acids aminoacid single linear protein unknown Human GDF-10 Protein 1..115 25 LysAla Arg Arg Lys Gln Trp Asp Glu Pro Arg Val Cys Ser Arg Arg 1 5 10 15Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Asn Glu Trp Ile Ile 20 25 30Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly Ala Cys Glu Phe 35 40 45Pro Met Pro Lys Ile Val Arg Pro Ser Asn His Ala Thr Ile Gln Ser 50 55 60Ile Val Arg Ala Val Gly Ile Ile Pro Gly Ile Pro Glu Pro Cys Cys 65 70 7580 Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu Phe Leu Asp Glu Asn 85 9095 Arg Asn Val Val Leu Lys Val Tyr Pro Asn Met Ser Val Asp Thr Cys 100105 110 Ala Cys Arg 115 115 amino acids amino acid single linear proteinunknown Murine GDF-10 Protein 1..115 26 Lys Ala Arg Arg Lys Gln Trp AspGlu Pro Arg Val Cys Ser Arg Arg 1 5 10 15 Tyr Leu Lys Val Asp Phe AlaAsp Ile Gly Trp Asn Glu Trp Ile Ile 20 25 30 Ser Pro Lys Ser Phe Asp AlaTyr Tyr Cys Ala Gly Ala Cys Glu Phe 35 40 45 Pro Met Pro Lys Ile Val ArgPro Ser Asn His Ala Thr Ile Gln Ser 50 55 60 Ile Val Arg Ala Val Gly IleVal Pro Gly Ile Pro Glu Pro Cys Cys 65 70 75 80 Val Pro Asp Lys Met AsnSer Leu Gly Val Leu Phe Leu Asp Glu Asn 85 90 95 Arg Asn Ala Val Leu LysVal Tyr Pro Asn Met Ser Val Glu Thr Cys 100 105 110 Ala Cys Arg 115

What is claimed is:
 1. Substantially pure growth differentiationfactor-10 (GDF-10) having the amino acid sequence as set forth in SEQ IDNO:5 or SEQ ID NO:25.
 2. An isolated polynucleotide encoding GDF-10polypeptide having the amino acid sequence as set forth in SEQ ID NO:5or SEQ ID NO:25.
 3. The polynucleotide of claim 2, wherein thepolynucleotide is isolated from a mammalian cell.
 4. The polynucleotideof claim 3, wherein the mammalian cell is selected from the groupconsisting of a mouse, rat, and human cell.
 5. An expression vectorcomprising the polynucleotide of claim
 2. 6. The vector of claim 5,wherein the vector is a plasmid.
 7. The vector of claim 5, wherein thevector is a viral vector.
 8. A host cell containing the vector of claim5.
 9. The host cell of claim 8, wherein the cell is prokaryotic.
 10. Thehost cell of claim 8, wherein the cell is eukaryotic.
 11. An isolatedpolynucleotide selected from the group consisting of: a) SEQ ID NO:4; b)SEQ ID NO:4, wherein T can also be U; c) nucleic acid sequencescomplementary to SEQ ID NO:4 and d) fragments of a), b), or c) that areat least 15 bases in length and that will hybridize to genomic DNA whichencodes the GDF-10 protein of SEQ ID NO:5 or SEQ ID NO: 25.